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pH-induced conformational changes in Clostridium difficile toxin B
M Qa'Dan1, L M Spyres, J D Ballard
1Department of Botany and Microbiology, The University of Oklahoma, Norman, Oklahoma 73019, USA.
Abstract:
Toxin B from Clostridium difficile is a monoglucosylating toxin that targets substrates within the cytosol of mammalian cells. In this study, we investigated the impact of acidic pH on cytosolic entry and structural changes within toxin B. Bafilomycin A1 was used to block endosomal acidification and subsequent toxin B translocation. Cytopathic effects could be completely blocked by addition of bafilomycin A1 up to 20 min following toxin treatment. Furthermore, providing a low extracellular pH could circumvent the effect of bafilomycin A1 and other lysosomotropic agents. Acid pH-induced structural changes were monitored by using the fluorescent probe 2-(p-toluidinyl) naphthalene-6-sulfonic acid, sodium salt (TNS), inherent tryptophan fluorescence, and relative susceptibility to a specific protease. As the toxin was exposed to lower pH there was an increase in TNS fluorescence, suggesting the exposure of hydrophobic domains by toxin B. The change in hydrophobicity appeared to be reversible, since returning the pH to neutrality abrogated TNS fluorescence. Furthermore, tryptophan fluorescence was quenched at the acidic pH, indicating that domains may have been moving into more aqueous environments. Toxin B also demonstrated variable susceptibility to Staphylococcus aureus V8 protease at neutral and acidic pH, further suggesting pH-induced structural changes in this protein.
Insights
Acidic pH triggers structural changes in Clostridium difficile Toxin B, affecting its cytosolic entry. This pH-induced hydrophobicity is reversible and impacts toxin activity, offering insights into its cellular mechanisms.
Area of Science:
- Microbiology
- Cell Biology
- Toxicology
Background:
- Clostridium difficile Toxin B is a key virulence factor.
- It functions as a monoglucosylating toxin targeting intracellular host proteins.
- Understanding toxin entry and structural dynamics is crucial for therapeutic development.
Purpose of the Study:
- To investigate the influence of acidic pH on Clostridium difficile Toxin B's cytosolic entry.
- To characterize pH-induced structural modifications in Toxin B.
- To explore the reversibility of these structural changes and their functional implications.
Main Methods:
- Utilized bafilomycin A1 to inhibit endosomal acidification and toxin translocation.
- Manipulated extracellular pH to assess its effect on toxin entry and cellular damage.
- Employed fluorescent probes (TNS) and intrinsic tryptophan fluorescence to monitor structural changes.
- Assessed protease susceptibility (Staphylococcus aureus V8 protease) at different pH levels.
Main Results:
- Bafilomycin A1 effectively blocked cytopathic effects of Toxin B.
- Low extracellular pH bypassed bafilomycin A1 inhibition, facilitating toxin entry.
- Acidic pH induced increased hydrophobicity and altered tryptophan fluorescence in Toxin B.
- These pH-induced structural changes were reversible upon returning to neutral pH.
- Toxin B exhibited altered protease susceptibility at acidic pH.
Conclusions:
- Acidic pH significantly impacts Clostridium difficile Toxin B's structure and cellular translocation.
- The observed changes in hydrophobicity and conformation are reversible.
- These findings provide insights into the mechanism of Toxin B entry into mammalian cells.